title: Characterization of a (D)-stereoselective aminopeptidase (DamA) exhibiting aminolytic activity and halophilicity from Aspergillus oryzae.
abstract: β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide (pNA)], the enzyme preferred D-Leu-pNA and D-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward D-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0-11.0. DamA also exhibited aminolytic activity, producing D-Leu-D-Leu-NH2 from D-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from D-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a D-amino acid at the N-terminus as well as physiologically active peptides.
authors.affiliation: National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Matsushita-Morita
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Sawaki
authors.initials: S
authors.lastname: Tada
authors.forename: Junichiro
authors.initials: J
authors.lastname: Marui
authors.forename: Ryota
authors.initials: R
authors.lastname: Hattori
authors.forename: Satoshi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Youhei
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Amano
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ishida
authors.forename: Michio
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Kusumoto
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology*; D043384:Glutamyl Aminopeptidase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D012965:Sodium Chloride; D043384:Glutamyl Aminopeptidase